The twin-arginine translocation (Tat) system.

In this Quick guide, Palmer and Berks introduce the twin-arginine translocation (Tat) systems. Tats are found in a variety of microbes and microbe-derived organelles, and are known to translocate folded substrate proteins across biological membranes.

Structural insights into the mechanism of protein transport by the Type 9 Secretion System translocon.

Secretion systems are protein export machines that enable bacteria to exploit their environment through the release of protein effectors. The Type 9 Secretion System (T9SS) is responsible for protein export across the outer membrane (OM) of bacteria of the phylum Bacteroidota. Here we trap the T9SS of Flavobacterium johnsoniae in the process of substrate transport by disrupting the T9SS motor complex. Cryo-EM analysis of purified substrate-bound T9SS translocons reveals an extended translocon structure in which the previously described translocon core is augmented by a periplasmic structure incorporating the proteins SprE, PorD and a homologue of the canonical periplasmic chaperone Skp. Substrate proteins bind to the extracellular loops of a carrier protein within the translocon pore. As transport intermediates accumulate on the translocon when energetic input is removed, we deduce that release of the substrate-carrier protein complex from the translocon is the energy-requiring step in T9SS transport.

Aberrant Topologies of Bacterial Membrane Proteins Revealed by High Sensitivity Fluorescence Labelling.

The cytoplasmic membrane compartmentalises the bacterial cell into cytoplasm and periplasm. Proteins located in this membrane have a defined topology that is established during their biogenesis. However, the accuracy of this fundamental biosynthetic process is unknown. We developed compartment-specific fluorescence labelling methods with up to single-molecule sensitivity. Application of these methods to the single and multi-spanning membrane proteins of the Tat protein transport system revealed rare topogenesis errors. This methodology also detected low level soluble protein mislocalization from the cytoplasm to the periplasm. This study shows that it is possible to uncover rare errors in protein localization by leveraging the high sensitivity of fluorescence methods.

New insights into the Tat protein transport cycle from characterizing the assembled Tat translocon.

The twin-arginine protein translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of chloroplasts. The Tat translocation site is transiently assembled by the recruitment of multiple TatA proteins to a substrate-activated TatBC receptor complex in a process requiring the protonmotive force. The ephemeral nature of the Tat translocation site has so far precluded its isolation. We now report that detergent solubilization of membranes during active transport allows the recovery of receptor complexes that are associated with elevated levels of TatA. We apply this biochemical analysis in combination with live cell fluorescence imaging to Tat systems trapped in the assembled state. We resolve sub-steps in the Tat translocation cycle and infer that TatA assembly precedes the functional interaction of TatA with a polar cluster site on TatC. We observe that dissipation of the protonmotive force releases TatA oligomers from the assembled translocation site demonstrating that the stability of the TatA oligomer does not depend on binding to the receptor complex and implying that the TatA oligomer is assembled at the periphery of the receptor complex. This work provides new insight into the Tat transport cycle and advances efforts to isolate the active Tat translocon.

Structures of the Type IX Secretion/Gliding Motility Motor from across the Phylum Bacteroidetes.

Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are energized by the GldLM motor complex, which transduces the proton motive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9-Å resolution (R. Hennell James, J. C. Deme, A. Kjaer, F. Alcock, et al., Nat Microbiol 6:221-233, 2021, https://dx.doi.org/10.1038/s41564-020-00823-6). Here, we present structures of homologous complexes from a range of pathogenic and environmental Bacteroidetes species at up to 3.0-Å resolution. These structures show that the architecture of the GldLM motor core is conserved across the Bacteroidetes phylum, although there are species-specific differences at the N terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility. IMPORTANCE Many bacteria in the Bacteroidetes phylum use the type IX secretion system to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex, which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species, including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organization of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the type IX secretion system, which is a virulence determinant in human and animal diseases.

The DNA transporter ComEC has metal-dependent nuclease activity that is important for natural transformation.

In the process of natural transformation bacteria import extracellular DNA molecules for integration into their genome. One strand of the incoming DNA molecule is degraded, whereas the remaining strand is transported across the cytoplasmic membrane. The DNA transport channel is provided by the protein ComEC. Many ComEC proteins have an extracellular C-terminal domain (CTD) with homology to the metallo-ß-lactamase fold. Here we show that this CTD binds Mn2+ ions and exhibits Mn2+ -dependent phosphodiesterase and nuclease activities. Inactivation of the enzymatic activity of the CTD severely inhibits natural transformation in Bacillus subtilis. These data suggest that the ComEC CTD is a nuclease responsible for degrading the nontransforming DNA strand during natural transformation and that this process is important for efficient DNA import.

Structure and mechanism of the proton-driven motor that powers type 9 secretion and gliding motility.

Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor and a proton-driven motor that powers gliding motility and the type 9 protein secretion system in Bacteroidetes bacteria. Here, we present cryo-electron microscopy structures of the gliding motility/type 9 protein secretion system motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identify protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane.

Author Correction: Structures of the stator complex that drives rotation of the bacterial flagellum.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structures of the stator complex that drives rotation of the bacterial flagellum.

The bacterial flagellum is the prototypical protein nanomachine and comprises a rotating helical propeller attached to a membrane-embedded motor complex. The motor consists of a central rotor surrounded by stator units that couple ion flow across the cytoplasmic membrane to generate torque. Here, we present the structures of the stator complexes from Clostridium sporogenes, Bacillus subtilis and Vibrio mimicus, allowing interpretation of the extensive body of data on stator mechanism. The structures reveal an unexpected asymmetric A5B2 subunit assembly where the five A subunits enclose the two B subunits. Comparison to structures of other ion-driven motors indicates that this A5B2 architecture is fundamental to bacterial systems that couple energy from ion flow to generate mechanical work at a distance and suggests that such events involve rotation in the motor structures.

Type 9 secretion system structures reveal a new protein transport mechanism.

The type 9 secretion system (T9SS) is the protein export pathway of bacteria of the Gram-negative Fibrobacteres-Chlorobi-Bacteroidetes superphylum and is an essential determinant of pathogenicity in severe periodontal disease. The central element of the T9SS is a so-far uncharacterized protein-conducting translocon located in the bacterial outer membrane. Here, using cryo-electron microscopy, we provide structural evidence that the translocon is the T9SS protein SprA. SprA forms an extremely large (36-strand) single polypeptide transmembrane ß-barrel. The barrel pore is capped on the extracellular end, but has a lateral opening to the external membrane surface. Structures of SprA bound to different components of the T9SS show that partner proteins control access to the lateral opening and to the periplasmic end of the pore. Our results identify a protein transporter with a distinctive architecture that uses an alternating access mechanism in which the two ends of the protein-conducting channel are open at different times.

Precursor-Receptor Interactions in the Twin Arginine Protein Transport Pathway Probed with a New Receptor Complex Preparation.

The twin arginine translocation (Tat) system moves folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Signal peptide-bearing substrates of the Tat pathway (precursor proteins) are recognized at the membrane by the TatBC receptor complex. The only established preparation of the TatBC complex uses the detergent digitonin, rendering it unsuitable for biophysical analysis. Here we show that the detergent glyco-diosgenin (GDN) can be used in place of digitonin to isolate homogeneous TatBC complexes that bind precursor proteins with physiological specificity. We use this new preparation to quantitatively characterize TatBC-precursor interactions in a fully defined system. Additionally, we show that the GDN-solubilized TatBC complex co-purifies with substantial quantities of phospholipids.

In vivo experiments do not support the charge zipper model for Tat translocase assembly.

The twin-arginine translocase (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat translocation site is formed by substrate-triggered oligomerization of the protein TatA. Walther and co-workers have proposed a structural model for the TatA oligomer in which TatA monomers self-assemble using electrostatic 'charge zippers' (Cell (2013) 132: 15945). This model was supported by in vitro analysis of the oligomeric state of TatA variants containing charge-inverting substitutions. Here we have used live cell assays of TatA assembly and function in Escherichia coli to re-assess the roles of the charged residues of TatA. Our results do not support the charge zipper model. Instead, we observe that substitutions of charged residues located in the TatA amphipathic helix lock TatA in an assembled state, suggesting that these charged residues play a critical role in the protein translocation step that follows TatA assembly.

Intermediates in the Sox sulfur oxidation pathway are bound to a sulfane conjugate of the carrier protein SoxYZ.

The Sox pathway found in many sulfur bacteria oxidizes thiosulfate to sulfate. Pathway intermediates are covalently bound to a cysteine residue in the carrier protein SoxYZ. We have used biochemical complementation by SoxYZ-conjugates to probe the identity of the intermediates in the Sox pathway. We find that unconjugated SoxYZ and SoxYZ-S-sulfonate are unlikely to be intermediates during normal turnover in disagreement with current models. By contrast, conjugates with multiple sulfane atoms are readily metabolised by the Sox pathway. The most parsimonious interpretation of these data is that the true carrier species in the Sox pathway is a SoxYZ-S-sulfane adduct.

A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase.

The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA-YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.

Assembling the Tat protein translocase.

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.

Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle.

The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a "suicide enzyme" strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein-protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway.

The TatC component of the twin-arginine protein translocase functions as an obligate oligomer.

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate-bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild-type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue-native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild-type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate-induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.

Mechanism of thiosulfate oxidation in the SoxA family of cysteine-ligated cytochromes.

Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.

The twin-arginine protein translocation pathway.

The twin-arginine translocation (Tat) system, found in prokaryotes, chloroplasts, and some mitochondria, allows folded proteins to be moved across membranes. How this transport is achieved without significant ion leakage is an intriguing mechanistic question. Tat transport is mediated by complexes formed from small integral membrane proteins from just two protein families. Atomic-resolution structures have recently been determined for representatives of both these protein families, providing the first molecular-level glimpse of the Tat machinery. I review our current understanding of the mechanism of Tat transport in light of these new structural data.